A large number of human and other mammalian proteins, including, for example, human growth hormone, human protein C clotting Factor VII and IL-18BP have been produced in host cells by transfecting these cells with DNA encoding these proteins and growing the recombinant cells under conditions favourable for the expression of the protein. Recombinant proteins can be produced also by transgenic animals and secreted into the milk. The recombinant proteins are secreted by the cells into the cell culture medium, into the milk or are present in cell lysates and must be separated from other cell components, such as cell waste products, cell debris and proteins or other collected material. Protein purification usually requires some type of chromatography separation (see review by Constans 2002).
The following chromatographic separations are widely used: gel filtration (GF), ion exchange (IEX), hydrophobic interaction (HI) chromatography, affinity chromatography and HPLC (high-performance liquid chromatography).
Protein purification generally takes place in three phases: a capture step, in which the desired protein is separated from other cellular components such as DNA and RNA; an intermediate step, in which proteins are isolated from contaminants similar in size or other physical/chemical properties; and finally a polishing step. Each purification stage have certain chromatography techniques and bead sizes that are best suited to the specific protein being purified.
The initial capture step typically involves protein isolation from a crude cell lysate or from cell culture medium and requires a resin with a high capacity and high flow rate. “Fast flow” resins with a large bead size and large bead size range (the range can vary widely from the average bead size) are suitable for this purpose.
Immunoglobulin (Ig) is defined as any of the structural cell antigen receptors; it is divided into five classes (IgM, IgG, IgA, IgD and IgE) on the basis of structure and biologic activity. The basic structural unit of the immunoglobulin molecule, referred to as a monomer, is a Y-shaped molecule composed of two heavy (H) chains having four domains each: one variable VH and three constant CH domains) and two light (L) chains having two domains each: one variable VL and one constant CL domain. VH and VL make up the antigen-binding site. The basic pattern of the immunoglobulin domains consists of two antiparallel, twisted β-sheets that surround an internal volume tightly packed with hydrophobic side chains (i.e. hydrophobic core). Depending on the degree of curvature of the sheets, the overall shape of the domain can be described either as a cylinder (β-barrel) or, if the two layers are straight, as a sandwich-like structure. The two β-sheets are covalently linked by a strongly but not rigorously conserved intra-chain disulfide bridge (Encyclopedia of Immunology Eds Roitt and Delves 1992, p92-93 and p476-477).
Ig-like domains have been identified in proteins from various kingdoms including eukaryotes and prokaryotes, including virus fungi and plants (Halaby et al. 1998). Ig-like domains are found in many proteins for example, in the bacterial enzymes β-galactosidase and chitinase A, in human receptors such as the growth hormone receptor, in cytokine receptors such as the IL-1 receptor (McMahan et al 1991), IL-6 receptor (Vollmer et al 1999) and human tissue factor (HFT) receptor (Halaby et al. 1999), in thyrosine kinase receptors that transduce growth factor dependent signals to the intracellular environment (Wiesmann et al. 2000), in immunoglobulin related proteins such as CD4, and in extracellular matrix proteins such as Fibronectin type III (Halaby et al. 1999).
Typically, Ig-like domains are composed of 7-10 β-strands, distributed between two sheets with specific topology and connectivity. Fifty-two 3D structures of Ig-like domains covering the immunoglobulin fold family (IgFF) were compared (Halaby et al. 1999) and the results show that most of the Ig-like domains display less than 10% sequence identity and that in the Ig-like domains most of the residues constituting the common core are hydrophobic. Thus, Ig-like domains have more structural than sequence similarities. The hydrophobic core has a major impact on the uniqueness and stability of the Ig fold. Despite the wide sequence variations in Ig-like domains, the maintenance of the Ig-fold seems to be enhanced by a conserved geometry of hydrogen bonds. Some proteins have more than one Ig-like domain, for example the mature type II IL-1 receptor has three immunoglobulin-like domains (McMahan et al. 1991) and the adhesion molecule VCAM has 7 Ig-like domains (Osborn et al. 1994).
The following are examples of important proteins having Ig-like domains: adhesion molecules such as NCAM (5 Ig-like domains), Fibronectin type III, ICAM-1, mad CAM-1, PE CAM-1, VCAM-1, titin and cadherin, neurocan, extracellular domains of cytokine receptors such as LIFR, CNTFR, IL-3R, IL5R, IL-6R, IL-12R, GM-CSFR and OSMR, growth factor receptors such as Vascular endothelial growth factor (VEGF) receptor (7 Ig-like domains), fibroblast growth factor (FGF) receptor, human platlet-derived growth factor (hPDGF) receptor, immune related receptors such as T cell receptor, major histocompatibility complex (MHC) proteins, macrophage colony stimulatory factor 1 receptor (CSF-1R), microglobulin-β, CTLA4 a receptor in T cells for B7 molecules (two Ig-like domains), B7 a B cell activation agent which regulates T cell proliferation and others such as neuregulin, coagulation factor XIII, NF-kB, superoxide dismutase and IL-18 binding protein.
Cytokine binding proteins usually consist of the extracellular ligand binding domains of their respective cell surface cytokine receptors (soluble cytokine receptors). The soluble receptors are produced either by alternative splicing or by proteolytic cleavage of the cell surface receptor. These soluble receptors have been described in the past: for example, the soluble receptors for IL-6 and IFN-γ (Novick et al. 1989), TNF (Engelmann et al. 1989 and Engelmann et al. 1990), IL-1 and IL-4 (Maliszewski et al. 1990) and IFN-α/β (Novick et al. 1994, Novick et al. 1992). One cytokine-binding protein, named osteoprotegerin (OPG, also known as osteoclast inhibitory factor—OCIF), a member of the TNFR/Fas family, appears to be the first example of a soluble receptor that exists only as a secreted protein (Anderson et al. 1997, Simonet et al. 1997, Yasuda et al. 1998).
An interleukin-18 binding protein (IL-18BP) which abolishes IL-18 induction of IFN-γ and IL-18 activation of NF-kB in vitro is known (Novick et al. 1999). IL-18BP is a soluble receptor that exist only as a secreted protein. IL-18BP has a single Ig-like domain and resembles the extracellular segment of cytokine receptors comprising Ig-like domains.
Another non-immunoglobulin protein comprising an Ig-like domain is the receptor for interleukin-6 (IL-6R). In the literature interleukin-6 has been proposed to act both as pro- and anti-inflammatory cytokine (reviewed in Heinrich et al., 1998, Jones et al. 2001 and Pedersen et al. 2001). The receptor complex mediating the biological activities of IL-6 consist of two distinct membrane bound glycoproteins, an 80 kDa cognate receptor subunit (IL-6R) and a 130 kDa signal-transducing element (gp130, CD130). Expression of gp130 is ubiquitous, in contrast, cellular distribution of IL-6R is limited and is predominantly confined to hepatocytes and leukocyte subpopulations. In addition to the membrane bound receptor, a soluble form of the IL-6R (sIL-6R) has been purified from human serum and urine. This soluble receptor binds IL-6 and prolongs its plasma half-life. More importantly the sIL-6R/IL-6 complex is capable of activating cells via interaction with gp130. This feature makes the sIL-6R/IL-6 complex an agonist for cell types that although they express gp130, would not inherently respond to IL-6 alone. Hence, the sIL-6R has the ability to widen the repertoire of cell types that are responsible to IL-6.
By fusing the entire coding regions of the cDNAs encoding the soluble IL-6 receptor (sIL-6R) and IL-6, a recombinant IL6-IL6R chimera was produced in human cells (Chebath et al. 1997). This IL6-IL6R chimera has enhanced IL-6-type biological activities and it binds with a much higher efficiency to the gp130 chain in vitro than does the mixture of IL-6 with sIL-6R (Kollet et al. 1999).
Mercapto-ethyl-pyridine (MEP) HYPERCEL® (BioSepra) is a Hydrophobic Charge Induction Chromatography (HCIC) resin. This resin was specifically designed to capture immunoglobulins (Boschetti 2000 and Life technologies Inc. 2000). At neutral pH, hydrophobic capture occurs in HCIC resin by both an aliphatic-hydrophobic spacer and a neutral (uncharged) pyridine ring. In contrast to HI chromatography, adsorption of antibodies from cell culture supernatants on HCIC resin is accomplished without the need of any pH or ionic strength adjustment. Once the pH is lowered from pH 7.2 to pH 4, the pyridine ring in the resin and the bound antibody become positively charged, due to charge repulsion, the immunoglobulins detaches and elutes from the column. Although this chromatography method is used for the capture of immunoglobulin, it could not be predicted that it would work for the capture of non-immunoglobulin proteins having an IgG-like domain, since the immunoglobulins have a distinctive sequences and moreover since the IgG-like domain in 52 different non-immunoglobulin proteins has less than 10% sequence identity (Halaby et al. 1999).
The present invention relates to a method for purifying non-immunoglobulin proteins having Ig-like domains from a biological fluid.